This invention relates to substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones, which show an inhibitory effect on hormone-sensitive lipase (HSL), and their pharmaceutically acceptable salts or acid addition salts. The present invention further relates to processes for the preparation of 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones, to the use of 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones and their pharmaceutically acceptable salts or acid addition salts as pharmaceuticals, including their use as inhibitors of HSL, and to pharmaceutical compositions comprising 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones and their pharmaceutically acceptable salts or acid addition salts, including their use in the treatment of non-insulin dependent diabetes mellitus and diabetic syndrome.
Certain 5-alkoxy-1,3,4-oxadiazol-2-ones substituted with an ortho-substituted phenyl ring or with fused-on five- or six-membered rings have anthelmintic (DE-A 26 04 110) and insecticidal effects (DE-A 26 03 877, EP-B 0 048 040, EP-B 0 067 471).
Certain 5-phenoxy-1,3,4-oxadiazol-2-ones with an ortho-substituted phenyl ring as substituents show an endoparasiticidal effect (EP-A 0 419 918).
An aim of the invention was to find compounds which show an inhibitory effect on hormone-sensitive lipase, HSL.
The present invention relates to substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones of the formula 1: 
in which:
R1 is C1-C6-alkyl, C3-C9-cycloalkyl, wherein both groups are optionally substituted one or more times by phenyl, C1-C4-alkyloxy, Sxe2x80x94C1xe2x80x94C4-alkyl, N(C1-C4-alkyl)2, and wherein phenyl is optionally substituted one or more times by halogen, C1-C4-alkyl, C1-C4-alkyloxy, nitro, CF3; and
R2, R3, R4 and R5 independently of one another are hydrogen, halogen, nitro, C1-C4-alkyl, C1-C9-alkyloxy; C6-C10-aryl-C1-C4-alkyloxy, C6-C10-aryloxy, C6-C10-aryl, C3-C8-cycloalkyl or Oxe2x80x94C3-C8-cycloalkyl, each of which is optionally substituted once, twice or three times by halogen, CF3, C1-C4-alkyloxy or C1-C4-alkyl; 2-oxopyrrolidin-1-yl, 2,5-dimethylpyrrol-1-yl or NR6-A-R7, with the proviso that R2, R3, R4 and R5 are not simultaneously hydrogen, and at least one of the radicals R2, R3, R4 or R5 is the radical 2-oxopyrrolidin-1-yl, 2,5-dimethylpyrrol-1-yl, or NR6-A-R7, and wherein:
R6 is hydrogen, C1-C4-alkyl or C6-C10-aryl-C1-C4-alkyl, wherein aryl may be substituted by halogen, CF3, C1-C8-alkyloxy or C1-C4-alkyl;
A is a single bond, COn, SOn, or CONH;
n is 1 or 2;
R7 is hydrogen, C1-C18-alkyl or C2-C18-alkenyl, wherein C1-C18-alkyl or C2-C18alkenyl are optionally substituted one to three times by C1-C4-alkyl, halogen, CF3, C1-C4-alkyloxy, N(C1-C4-alkyl)2, xe2x80x94COOH, C1-C4-alkyloxycarbonyl, C6-C12-aryl, C6-C12-aryloxy, C6-C12-arylcarbonyl, C6-C10-aryl-C1-C4-alkyloxy or oxo, wherein aryl is in turn optionally substituted by halogen, C1-C4-alkyl, aminosulfonyl or methylmercapto;
C6-C10-aryl-C1-C4-alkyl, C5-C8-cycloalkyl-C1-C4-alkyl, C5-C8-cycloalkyl, C6-C10-aryl-C2-C6-alkenyl, C6-C10-aryl, biphenylyl, biphenylyl-C1-C4-alkyl, indanyl, each of which is optionally substituted once or twice by C1-C18-alkyl, C1-C1 8-alkyloxy, C3-C8-cycloalkyl, COOH, hydroxyl, C1-C4-alkylcarbonyl, C6-C10-aryl-C1-C4-alkyl, C6-C10-aryl-C1-C4-alkyloxy, C6-C10-aryloxy, nitro, cyano, C6-C10-aryl, fluorosulfonyl, C1-C6-alkyloxycarbonyl, C6-C10-arylsulfonyloxy, pyridyl, NHSO2-C6-C10-aryl, halogen, CF3 or OCF3, wherein alkyl is in turn optionally substituted by C1-C4-alkyloxycarbonyl, CF3 or carboxyl, and aryl is also optionally substituted by halogen, CF3 or C1-C4-alkyloxy;
or the group Het-(CH2)rxe2x80x94,
wherein r=0, 1, 2 or 3 and Het=a saturated or unsaturated 5-7-membered heterocycle, optionally benzo-fused and optionally substituted by C1-C4-alkyl, C6-C10-aryl, halogen, C1-C4-alkyloxy, C1-C4-alkyloxycarbonyl, C6-C10-aryl-C1-C4-alkyl, C6-C10-aryl-C1-C4-alkylmercapto or nitro, wherein the benzo-fused aryl is in turn optionally substituted by halogen, C1-C4-alkyloxy or CF3 and the alkyl in arylalkyl is also optionally by methoxy and CF3,
and their pharmaceutically acceptable salts and acid addition salts.
Said aryl radicals are optionally substituted one or more times by C1-C9-alkyl, C1-C8-alkyloxy, halogen, and trifluoromethyl. Said cycloalkyl radicals are optionally substituted one or more times by C1-C4-alkyl, C6-C10-aryl, and said alkyl radicals are optionally substituted by hydroxyl, di-C1-C4-alkylamino and fluorine. Halogen is fluorine, chlorine, bromine, generally fluorine and chlorine. Alkyl, alkenyl, alkyloxy, etc. are branched or unbranched. The phrase xe2x80x9cis optionally substitutedxe2x80x9d means that the relevant group is or is not substituted.
Pharmaceutically acceptable salts of compounds of the formula 1 include their organic and inorganic salts, as described in Remington""s Pharmaceutical Sciences (A. R. Gennard Editor, Mack Publishing Co., Easton, Pa., USA, 17th Ed., p 1418, (1985)). Examples of acidic groups include, inter alia, sodium, potassium, calcium and ammonium salts. Examples of basic groups include, inter alia, salts of hydrochloric acid, sulfuric acid, phosphoric acid, or of carboxylic acids or sulfonic acids, such as, for example, acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid.
Typical compounds of the formula 1 are those in which:
R1 is C1-C4-alkyl; and/or
R5 is hydrogen; and/or
R2 is hydrogen, halogen, C1-C4-alkyl, C1-C9-alkyloxy or amino.
Further examples of compounds of the formula 1 are those in which:
R3 is hydrogen, C1-C4-alkyl, C6-C10-aryl-C1-C4-alkyloxy, which is optionally substituted in the aryl moiety by halogen, or is NR6-A-R7 wherein
R6=hydrogen or benzyl,
A=single bond and
R7=C6-C10-aryl-C1-C4-alkyl, which is optionally substituted by halogen, CF3, cyano, phenyl-C1-C4-alkyloxy, CF3-phenoxy, C5-C8-cycloalkyl or fluorosulfonyloxy;
C1-C12-alkyl, which is optionally substituted by C1-C4-alkyloxy, phenyl, CF3 or phenyl-C1-C4-alkyloxy; C2-C12-alkenyl;
or the group Het-(CH2)rxe2x80x94,
wherein r=0 or 1, and Het=a saturated or unsaturated 5-7-membered heterocycle, which is optionally benzo-fused and optionally substituted by C1-C4-alkyl or halogen.
Additional compounds of the formula 1 are those in which:
R4 is hydrogen, 2-oxopyrrolidin-1-yl, 2,5-dimethylpyrrol-1-yl or C6-C10-aryl-C1-C4-alkyloxy, which is optionally substituted by halogen, and/or: compounds of the formula 1 in which: R4 is NR6-A-R7, wherein
R6=hydrogen or methyl,
A=single bond and
R7=hydrogen;
C1-C12-alky, which is optionally substituted once or twice by halogen;
C2-C18-alkenyl, which is optionally substituted once or twice by C1-C4-alkyl or C1-C4-alkyloxycarbonyl;
C6-C10-aryl-C1-C4-alky, which is optionally substituted by halogen, C1-C6-alkyloxy, CF3, cyano, C5-C6-cycloalkyl, C1-C4-alkyloxycarbonyl, C6-C10-aryl-C1-C4-alkyl, C6-C10-aryl-C1-C4-alkyloxy, wherein aryl is further optionally substituted by halogen or CF3;
C5-C8-cycloalkyl-C1-C4-alkyl;
or the group Het-(CH2)rxe2x80x94,
wherein r=1, 2 or 3 and Het=a saturated or unsaturated 5-7-membered heterocycle, which is optionally substituted by halogen, C1-C4-alkyloxy or C1-C4-alkyloxycarbonyl, and/or compounds of the formula 1 in which:
R4 is NR6-A-R7, wherein
R6=hydrogen,
A=xe2x80x94COxe2x80x94 and
R7=C1-C18-alkyl, which is optionally substituted by halogen, phenyl, phenoxy, phenylcarbonyl or C1-C4-alkyloxycarbonyl, wherein phenoxy is optionally substituted by methyl, halogen or methylmercapto;
C2-C18-alkenyl, which is optionally substituted by C6-C10-aryl;
C6-C10-aryl, which is optionally substituted by halogen, C1-C8-alkyl, phenyl-C1-C4-alkyl, CF3, OCF3, fluorosulfonyl, C1-C4-alkyloxycarbonyl, phenoxy, wherein aryl is optionally substituted by C1-C4-alkyloxy;
C6-C10-aryl-C1-C4-alkyl, wherein alkyl is optionally substituted by methoxy or CF3 and aryl by halogen;
or the group Het-(CH2)rxe2x80x94,
wherein r=0 and Het=a saturated or unsaturated 5-7-membered heterocycle, which is optionally benzo-fused and optionally substituted by C1-C4-alkyl, halogen, C1-C4-alkyloxy, halophenyl or halobenzylmercapto, wherein benzo-fused aryl is optionally substituted by halogen or methoxy, and/or compounds of the formula 1 in which:
R4 is NR6-A-R7, wherein
R6=hydrogen,
A=xe2x80x94CO2xe2x80x94 and
R7=C1-C18-alkyl, which is substituted by CF3 or phenyl;
C6-C10-aryl;
C6-C10-aryl-C1-C4-alkyl, which is substituted by C1-C4-alkyl, halogen, CF3 or OCF3, benzyloxy or phenyl;
or the group Het-(CH2)rxe2x80x94,
wherein r=0 or 1 and Het=a saturated or unsaturated 5-7-membered heterocycle, which is optionally benzo-fused and optionally substituted by C1-C4-alkyl or benzyl, and/or compounds of the formula 1 in which:
R4 is NR6-A-R7, wherein
R6=hydrogen,
A=xe2x80x94SO2xe2x80x94 and
R7=C1-C6-alky, which is optionally substituted by CF3;
C2-C4-alkenyl, which is optionally substituted by phenyl;
C6-C10-aryl, which is optionally substituted by C1-C6-alkyl, halogen,
C1-C4-alkyloxy or benzyl;
biphenylyl-C1-C4-alkyl substituted by halogen;
or the group Het-(CH2)rxe2x80x94,
wherein r=0 and Het=a saturated or unsaturated 5-7-membered heterocycle, and/or compounds of the formula 1 in which:
R4 is NR6-A-R7, wherein
R6=hydrogen,
A=xe2x80x94COxe2x80x94NHxe2x80x94 and
R7=C1-C10-alkyl, which is optionally substituted by C1-C4-alkyloxycarbonyl, N(C1-C4-alkyl)2 or phenyl, which is in turn optionally substituted by halogen or aminosulfonyl;
C6-C10-aryl, which is optionally substituted by C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxycarbonyl, phenoxy, OCF3, benzyl or pyridyl, wherein alkyl is optionally substituted by C1-C4-alkyloxycarbonyl or carboxyl;
C5-C8-cycloalky, which is optionally substituted by hydroxyl, or indanyl;
or the group Het-(CH2)rxe2x80x94,
wherein r=0 or 1 and Het=a saturated or unsaturated 5-7-membered heterocycle, which is optionally substituted by benzyl.
Other typical compounds of the formula 1 are those in which R1 is methyl.
Representative compounds of the formula 1 are those mentioned in Examples 21, 22, 27, 28, 30 to 34, 36 to 42, 53, 54, 58, 60, 62, 65, 69, 71, 74, 92, 97,107,116,128, 130, 136,139,142,152,166 and 171.
The compounds of the invention have a surprising inhibitory effect on hormone-sensitive lipase, HSL, an allosteric enzyme in adipocytes, which is inhibited by insulin and is responsible for the breakdown of fats in fat cells and for the transfer of fat constituents into the blood stream. Inhibition of this enzyme thus corresponds to an insulin-like effect of the compounds of the invention, which eventually leads to a reduction of free fatty acids in the blood and of blood glucose. Therefore, the compounds of the invention can be employed in the treatment of metabolic disturbances such as, for example, non-insulin-dependent diabetes mellitus, diabetic syndrome, and direct damage to the pancreas.
The compounds of the invention can be prepared in various ways by methods known per se. 
For example, substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones of the formula 1 can be prepared by reacting hydrazines of the formula 2 with chloroformic esters of the formula 3 or other reactive carbonic ester derivatives, in which R1, R2, R3, R4 and R5 are as defined above, to give the compounds of the formula 4, which are acylated with phosgene, carbonyldiimidazole, diphosgene or triphosgene, cyclized and converted, where appropriate, by further chemical modification of the radicals R2-R5, such as, for example, by reduction of nitro to amino radicals by known processes, and subsequent acylation or alkylation, into compounds of the formula 1. Since acids are usually liberated in these reactions, neutralization is advisable by adding bases such as pyridine, triethylamine, sodium hydroxide solution or alkali metal carbonates. The reactions can be carried out in wide temperature ranges. It has proved advantageous to operate in the temperature range from 0xc2x0 C. to the boiling point of the solvent used. Examples of solvents employed are methylene chloride, THF, DMF, toluene, ethyl acetate, n-heptane, dioxane, and diethyl ether.
The hydrazines of the formula 2 can be prepared by known methods, for example by diazotization of the corresponding anilines and 
subsequent reduction by known methods or by nucleophilic substitution of suitably substituted phenyl derivatives of the formula 6 (X=F, Cl, Br, l, OSO2CF3) with hydrazine hydrate. Such suitable phenyl derivatives may be nitro-substituted halobenzenes, such as fluoro- and chloronitrobenzenes, from which the compounds of the invention can be prepared by known methods at a suitable point in the synthetic route by reduction and reaction with acylating or alkylating agents such as, for example, acid chlorides, anhydrides, isocyanates, chloroformic esters, sulfonyl chlorides or alkyl and arylalkyl halides, or by reductive alkylation with aldehydes.
The effect of the compounds of the invention on HSL was tested using the following enzyme assay system:
Enzyme Preparation:
Preparation of Partially Purified HSL:
Isolated rat fat cells were obtained from epididymal adipose tissue from untreated male rats (Wistar, 220-250 g) by collagenase treatment according to published methods (e.g., S. Nilsson et al., Anal. Biochem. 158:399-407 (1986); G. Fredrikson et al., J. Biol. Chem. 256:6311-6320 (1981); H. Tornquist et al., J. Biol. Chem. 251:813-819 (1976)). The fat cells from 10 rats were washed three times by flotation with 50 ml each time of homogenization buffer (25 ml tris/HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 10 xcexcg/ml leupeptin, 10 xcexcg/ml antipain, 20 xcexcg/ml pepstatin) and finally taken up in 10 ml of homogenization buffer. The fat cells were homogenized in a Teflon-in-glass homogenizer (Braun-Melsungen) by 10 strokes at 1500 rpm and 15xc2x0 C. The homogenate was centrifuged (Sorvall SM24 tubes, 5000 rpm, 10 min, 4xc2x0 C.). The subnatant between the fatty layer at the top and the pellet was removed and the centrifugation was repeated. The subnatant resulting therefrom was recentrifuged (Sorvall SM24 tubes, 20000 rpm, 45 min, 4xc2x0 C.). The subnatant was removed and mixed with 1 g of heparin-Sepharose (Pharmacia-Biotech, CL-6B, 5xc3x97washed with 25 mM tris/HCl, pH 7.4,150 mM NaCl). After the mixture had been incubated at 4xc2x0 C. for 60 min (shaking at 15-min intervals), it was centrifuged (Sorvall SM24 tubes, 3000 rpm, 10 min, 4xc2x0 C.). The supernatant was adjusted to pH 5.2 by adding glacial acetic acid and incubated at 4xc2x0 C. for 30 min. The precipitates were collected by centrifugation (Sorvall SS34, 12000 rpm, 10 min, 4xc2x0 C.) and suspended in 2.5 ml of 20 mM tris/HCl, pH 7.0,1 mM EDTA, 65 mM NaCl, 13% sucrose, 1 mM DTT, 10 xcexcg/ml leupeptin/pepstatin/antipain. The suspension was dialyzed against 25 mM tris/HCl, pH 7.4, 50% glycerol, 1 mM DTT, 10 xcexcg/ml leupeptin, pepstatin, antipain at 4xc2x0 C. overnight and then loaded onto a hydroxy apatite column (0.1 g per 1 ml of suspension, equilibrated with 10 mM potassium phosphate, pH 7.0, 30% glycerol, 1 mM DTT). The column was washed with four volumes of equilibration buffer at a flow rate of 20 to 30 ml/h. The HSL was eluted with one volume of equilibration buffer containing 0.5 M potassium phosphate, and then dialyzed (see above) and concentrated 5- to 10-fold by ultrafiltration (Amicon Diaflo PM 10 filter) at 4xc2x0 C. The partially purified HSL can be stored at xe2x88x9270xc2x0 C. for 4 to 6 weeks.
Assay:
To prepare the substrate, 25-50 xcexcCi of [3H]trioleoylglycerol (in toluene), 6.8 xcexcmol of unlabeled trioleoylglycerol and 0.6 mg of phospholipids (phosphatidylcholine/phosphatidylinositol 3:1 w/v) were mixed, dried with N2 and then taken up in 2 ml of 0.1 M KPi (pH 7.0) by ultrasonic treatment (Branson 250, microtip, setting 1-2, 2xc3x971 min at 1-min intervals). After addition of 1 ml of KPi and renewed ultrasonic treatment (4xc3x9730 sec on ice in 30-sec intervals), 1 ml of 20% BSA (in KPi) was added (final concentration of trioleoylglycerol 1.7 mM). For the reaction, 100 xcexcl of substrate solution were pipetted into 100 xcexcl of HSL solution (HSL prepared as above, diluted in 20 mM KPi, pH 7.0, 1 mM EDTA, 1 mM DTT, 0.02% BSA, 20 xcexcg/ml pepstatin, 10 xcexcg/ml leupeptin) and incubated at 37xc2x0 C. for 30 min. Addition of 3.25 ml of methanol/chloroform/heptane (10:9:7) and of 1.05 ml of 0.1 M K2CO3, 0.1 M boric acid (pH 10.5) was followed by thorough mixing and finally centrifugation (800xc3x97g, 20 min). After phase separation, one equivalent of the upper phase (1 ml) was removed and the radioactivity was determined by liquid scintillation measurement.
Evaluation:
Substances were normally tested in four independent mixtures. The inhibition of the HSL enzymatic activity by a test substance was determined by comparison with an uninhibited control reaction. The IC50 was calculated via an inhibition plot with at least 10 concentrations of the test substance. The data was analyzed using the software package GRAPHIT, Elsevier-BIOSOFT.
Selected compounds of the invention showed the following effect, as measured by this assay:
The following examples illustrate the preparation methods in detail without restricting them.